Use of lactobacillus for liver protection

ABSTRACT

A method for liver protection of a mammal is provided and includes administering an effective amount of isolated  Lactobacillus plantarum  CMU995 thereto. Providing a new use of  Lactobacillus plantarum  CMU995, which is deposited at the Food Industry Research and Development Institute (FIRDI) in Taiwan under accession number BCRC 910472 and in the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM 23780.

RELATED APPLICATIONS

The present application is a Divisional Application of the applicationSer. No. 13/736,089, filed Jan. 8, 2013, the entire contents of whichare hereby incorporated herein by reference, which claims priority toTaiwan Application Serial Number 101131368, filed Aug. 29, 2012, all ofwhich are herein incorporated by reference.

BACKGROUND

1. Field of Invention

The present disclosure relates to a use of a Lactobacillus. Moreparticularly, the present disclosure relates to a use of a Lactobacillusplantarum, strain CMU995, for liver protection.

2. Description of Related Art

Lactobacillus is one of the main bacteria existed in the intestinaltract of a human being or an animal. Because of the beneficial effectson the physiological activity of a human or an animal body,Lactobacillus is often added into various probiotic products. Forexample, Lactobacillus can inhibit the growth of enteric pathogens (suchas Salmonella or Escherichia) or resist the invasion of the pathogens,for instance, preventing the invasion of Salmonella typhimurium to thegastrointestinal epithelial cells. There are many patents related to theapplications of Lactobacillus for inhibiting the pathogens. For example,U.S. Pat. No. 5,603,930 discloses a Lactobacillus johnsonii that caninhibit enterotoxin and intestinal invasive pathogens; U.S. Pat. No.3,953,609 discloses a Lactobacillus lactis that can inhibit the growthof Escherichia in the digestive system; and U.S. Pat. No. 6,491,956discloses a Lactobacillus acidophilus that can prevent and treatgastritis, duodenal ulcer, and gastric ulcer caused by Helicobacterpylori infection.

Despite basic physiological/pharmacological activities, Lactobacillusmust have two important characteristics to function effectively withinthe animal body. First, the Lactobacillus must have strong resistance tothe gastric acid and choline secreted by the animal gastrointestinaltract to survive in the digestive system and reach to the intestinaltract to perform its function. Then, the Lactobacillus must be able tostrongly adhere to the intestinal epithelial cells of an animal host tocompete with other pathogens in the gastrointestinal tract and avoidbeing expelled by the pathogens. In addition, because the pathogens alsoadhere onto the intestinal epithelial cells and so infective to thehost, the Lactobacillus would be able to effectively expel the pathogensto protect the gastrointestinal tract from being infected if theLactobacillus has the even stronger adhesion ability to the intestinalepithelial cells.

The novel strain of Lactobacillus plantarum, which was first isolated bythe inventors of the present invention, has been proven that, apart fromthe ability of inhibiting the pathogens, it has excellent adhesionability to the cells of the gastrointestinal tract and the urinary tractthrough in vivo and in vitro experiments. Therefore, the Lactobacillusplantarum can effectively and durably protect the gastrointestinal tractand the urinary tract from pathogenic infections. It also been foundthat this strain can directly inhibit the growth of the pathogens toprevent and treat diseases caused by the pathogens.

SUMMARY

An aspect of the present disclosure is to provide a Lactobacillusplantarum, strain CMU995, namely Lactobacillus plantarum CMU995 in thefollowings, which is capable of preventing intestinal epithelial celldamages caused by intestinal endotoxin, thereby stabilizing theintestinal epithelial cells. In animal experiments, the Lactobacillusplantarum CMU995 is capable of inhibiting alcohol-induced endotoxinpassing through intestines and then invading into blood. Therefore, theLactobacillus plantarum CMU995 is able to inhibit alcohol or endotoxin-induced hepatitis or enteritis. It is worth to be mentioned that,results according to clinical experiments shows that the Lactobacillusplantarum CMU995 is also capable of decreasing levels of blood endotoxinand blood ammonia in patients with hepatocellular carcinoma or livercirrhosis. Thus, the Lactobacillus plantarum CMU995 can be applied totreatments and/or preventions of diseases caused by alcohol and/orendotoxin.

According to one embodiment of the present disclosure, a method forliver protection of a mammal includes administering an effective amountof isolated Lactobacillus plantarum CMU995 to the mammal. Theaforementioned Lactobacillus plantarum CMU995 is deposited at the FoodIndustry Research and Development Institute (FIRDI) in Taiwan underaccession number BCRC 910472 and in the German Collection ofMicroorganisms and Cell Cultures (DSMZ) under accession number DSM23780.

According to another embodiment of the present disclosure, a probioticcomposition for liver protection comprises powdered Lactobacillusplantarum CMU995 in which the concentration of said powderedLactobacillus plantarum CMU995 is 1×10⁵ CFU/g to 1×10¹² CFU/g.

It is to be understood that both the foregoing general description andthe following detailed description are by examples, and are intended toprovide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more fully understood by reading the followingdetailed description of the embodiment, with reference made to theaccompanying drawings as follows:

FIG. 1 is a bar chart illustrating different levels of AST (aspartateaminotransferase) in mice blood serum during an animal experimentaccording to example II of the present disclosure;

FIG. 2 is a bar chart illustrating different levels of ALT (alanineaminotransferase) in mice blood serum during an animal experimentaccording to example II of the present disclosure;

FIG. 3 is a bar chart illustrating different levels of triglyceride inmice blood serum during an animal experiment according to example II ofthe present disclosure;

FIG. 4 is a bar chart illustrating different levels of cholesterol inmice blood serum during an animal experiment according to example II ofthe present disclosure;

FIG. 5 is a bar chart illustrating different levels of endotoxin in miceblood serum during an animal experiment according to example II of thepresent disclosure;

FIG. 6 is a bar chart illustrating different levels of endotoxin in miceliver during an animal experiment according to example II of the presentdisclosure;

FIG. 7 is a bar chart illustrating different levels of cytokine TNF-α inmice liver during an animal experiment according to example II of thepresent disclosure;

FIG. 8 is a bar chart illustrating different levels of cytokine TNF-α inmice large intestine during an animal experiment according to example IIof the present disclosure;

FIG. 9 a is a bar chart illustrating different levels of cytokine TNF-αof the blood samples from cirrhosis patients being treated with theprobiotic composition according to a clinical trial of example II of thepresent disclosure;

FIG. 9 b is a bar chart illustrating different levels of blood ammoniaof the blood samples from cirrhosis patients being treated with theprobiotic composition according to a clinical trial of example II of thepresent disclosure; and

FIG. 9 c is a bar chart illustrating different levels of endotoxin, ofthe blood samples from cirrhosis patients being treated with theprobiotic composition according to a clinical trial of example II of thepresent disclosure;

DETAILED DESCRIPTION

The present disclosure can be understood more clearly with theadditional description which follows, which refers to non limitingexamples illustrating properties of a Lactobacillus plantarum strain,CMU995, named Lactobacillus plantarum CMU995 in the followings, inrelation to liver and/or intestinal protection.

A novel use of the Lactobacillus plantarum CMU995 is provided for liverprotection, in which said Lactobacillus plantarum CMU995 was screenedout and elected earlier by the inventor of the present invention herein,and the Lactobacillus plantarum CMU995 was not readily available to thepublic at the time of invention. The Lactobacillus plantarum CMU995 isdeposited at the Food Industry Research and Development Institute(FIRDI) in Taiwan, 331 Shih-Pin Road Hsinchu, 300 Taiwan R.O.C., on Apr.30, 2010, under accession number BCRC 910472, and in the GermanCollection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstraβe7B, 38124 Braunschweig, Germany, on Jul. 23, 2010, under accessionnumber DSM 23780. DSMZ has been accepted for deposit under the BudapestTreaty on the International Recognition of Deposit of Microorganisms forthe purpose of Patent Procedure. All restrictions on the availability tothe public of the material so deposited will be irrevocably removed uponthe granting of a patent.

Subsequent to deposit of strain DSM 23780, the taxonomic classificationof Lactobacillus plantarum CMU995 is as follows:

Taxonomy of Lactobacillus plantarum CMU995 DSM 23780 (Morphological andphysiological characteristics) Characteristics of colony Diameter ofcolony: 0.5~1 mm, (2 days, MSR Agar) white-gray, smooth Cell form andlength Short rod-shaped (MRS Bouillon) Adherent ability on the Stronggastrointestinal epithelial cells Lactic acid configuration D Growth at15° C. Positive Growth at 45° C. Positive End Ph in MRS Bouillon 3.5~4.2Gas formation out of glucose Positive Ammonia out of arginine NegativeDiamino pimelic acid Negative

According to earlier results of regardless in vitro whether in vivaexperiments, the aforementioned Lactobacillus plantarum CMU995 iscapable of inhibiting pathogen activity. Additionally, the Lactobacillusplantarum CMU995 is also capable of adhering to intestinal as well asurethral epithelial cells with great adhesion efficiency, so that directentries of pathogens into blood via intestine and urine can beprevented, thereby protecting intestine and urine.

Since alcohol commonly causes liver diseases, the Lactobacillusplantarum CMU995 is also able to prevent alcohol being intaken intoblood and reduces blood alcohol, thereby protecting the liver. Inaddition, the Lactobacillus plantarum CMU995 can be incorporated intopharmaceutical compositions, food, or drinks. Furthermore, because thatalcohol causes increments of intestinal pathogens and toxins, theLactobacillus plantarum CMU995 may also inhibit the increments ofintestinal pathogens and toxins as well, therefore further inhibitingtoxins or pathogens to be uptaken by liver or other organs. In short,the Lactobacillus plantarum CMU995 can be widely used for toxinexclusion, removal, and liver protection.

The Lactobacillus plantarum CMU995 and a probiotic compositioncontaining the Lactobacillus plantarum CMU995 is used for preventinghepatitis caused by alcohol and endotoxin, and reducingendotoxin-induced liver lesions and/or hepatocarcinogenesis. Theprobiotic composition can be supplied in a form of a pharmaceuticalcomposition, a feed, a drink a nutrient supplement, a diary, a food, ora healthy food. What is more, the aforementioned probiotic compositioncan be also provided in a form of a powder, a tablet, a granule, asuppository, a capsule, an ampoule, a liquid, or a spray. The probioticcomposition containing the Lactobacillus plantarum CMU995 is capable ofpreventing or treating various diseases, and can be widely used toprotect intestinal cells, reduce intestinal and blood toxin, and inhibitalcohol intake, thus protecting the liver.

EXAMPLES

The following examples are described for those skilled in the art tofurther understand the present disclosure and should not be limited tothe present disclosure.

I. Adhesion Analysis (1) Lactobacilli Preparation

Lactobacillus strains used in this cell adhesion test includesLactobacillus plantarum CMU995 of the present disclosure, Lactobacillusrhamnosus GG (LGG, ATCC 53103), Lactobacillus plantarum 299v, andLactobacillus casei Shirota. Each of these strains were activated twicein MRS broth (DIFCO®) before transferring to 5 ml MRS broth forculturing. After culturing for 24 hours, 1 ml of each strain werecentrifuged at 6,000 rpm for 10 minutes and then washed twice withphosphate buffer saline (PBS), pH 7.2. Then, concentrations of eachcultured strain were adjusted to 10⁹ CFU (colony-forming unit)/ml inaccordance with a value of OD₆₀₀ for further adhesion experiments.

(2) Cell Culture

Cell lines used herein are all kindly provided by the BioresourceCollection and Research Center, Taiwan, and include human colonadenocarcinoma (strain Caco-2 and HT-29), and gastric adenocarcinoma(strain AGS). The Caco-2 and HT-29 cells were cultured in DMEM cellculture medium with 10% fetal bovine serum, and the AGS cells werecultured in F-12 cell culture medium with 10% fetal bovine serum. Thesecells were all activated and stabilized before proceeding on furtherexperiments.

(3) Lactobacillus Adhesion Analysis

The activated AGS, Cacti-2, and HT-29 cells mentioned above were thenseeded in a 96-well culture plate, respectively; each well of the96-well culture plate containing 200 μl of corresponding culture mediawas seeded with about 10⁴ cells, and the culture media were beingreplaced with fresh media of the same composition every 24 hours. Afterculturing for 48 hours, 20 μl of each activated Lactobacillus plantarumCMU995, Lactobacillus rhamnosus GG (LGG, ATCC 53103) Lactobacillusplantarum 299v, and Lactobacillus casei Shirota were added to the cellsrespectively and incubated for 1 hour for adhesion, and then non-adheredLactobacilli were discarded by washing with PBS three times. In eachwell, cells and the Lactobacilli adhered thereto were fixed pith 10%formaldehyde for 30 minutes followed by wash with PBS three times.Subsequently, fixed Lactobacilli adhered cells were stained with crystalviolet for 5 minutes followed by wash with 75% ethanol three times.Adhesion efficiencies were acquired by counting total amounts of adheredLactobacilli onto 50 cells under randomized microscopic areas of aphase-contrast microscope; thereby an average amount of Lactobacillusadhering to each cell was calculated. The results are shown in Table 1.

TABLE 1 The amount of adhered Lactobabilli (CFU/cell) strains Caco-2HT-29 AGS Lactobacillus plantarum CMU995 53.6 ± 6.8 58.2 ± 10.5 51.7 ±6.3 LGG (ATCC 53103)  7.5 ± 1.5 6.7 ± 2.9  4.4 ± 0.8 Lactobacillus caseiShirota  3.2 ± 3.4 5.5 ± 2.1  2.8 ± 3.0 Lactobacillus plantarum 299v15.7 ± 3.5 16.5 ± 3.2  12.6 ± 2.4

As shown in Table 1, comparing to LGG (ATCC 53103), Lactobacillus caseiShirota, Lactobacillus plantarum 299v, obviously, the Lactobacillusplantarum CMU995 has significant adhesion ability to cell lines ofwhether strain Caco-2. HT-29, or AGS.

II. Animal Experiment (1) Animal Grouping

8-week old C57BL6J mice were randomly divided into 5 groups with 8 micein each group, and the initial weight of each mouse is 23±1 g. The 5groups include:

Group A; Control group: The mice were freely fed with sterilizedLieber-DeCarli liquid diet without ethanol added, and were fed by 0.2 mlsterilized physiological saline solution every day.

Group B; Negative control group: The mice were freely fed withethanol-containing sterilized Lieber-DeCarli liquid diet, and were fedby 0.2 sterilized physiological saline solution every day.

Group C; Lactobacillus plantarum CMU995 experimental group: The micewere freely fed with ethanol-containing sterilized Lieber-DeCarli liquiddiet, and were fed by 0.2 ml of 2×10¹⁰ CFU/ml Lactobacillus plantarumCMU995 every day.

Group D; Lactobacillus rhamnosus GG (LGG) experimental group: The micewere freely fed with ethanol-containing sterilized Lieber-DeCarli liquiddiet, and were fed by 0.2 ml of 2×10¹⁰ CFU/ml Lactobacillus rhamnosus GGevery day.

Group E; Lactobacillus casei Shirota (LC) experimental group: The micewere freely fed with ethanol-containing sterilized Lieber-DeCarli diet,and were fed by 0.2 ml of 2×10¹⁰ CFU/ml Lactobacillus casei Shirotaevery day.

(2) Lactobacilli Preparation

Each Lactobacillus casei Shirota, Lactobacillus plantarum 299v,Lactobacillus rhamnosus GG, and Lactobacillus plantarum CMU995 werelyophilized into powder with a concentration of 1×10¹¹ CFU/g, and wereall stored in 4° C. These lyophilized Lactobacillus powder were freshlyprepared before feeding experimental mice daily by re-suspending anddissolving them in sterilized physiological saline solution to a finalconcentration of 2×10¹⁰ CFU/ml.

(3) Lactobacilli Feeding and Hepatitis Induction

At the beginning of this experiment, mice of each group were freely fedwith Lieber-DeCarli liquid diet for a week. Subsequently, mice of eachgroup were started to be fed with 0.2 ml of different experimentalsamples daily; mice of group A and group B were fed with physiologicalsaline solution daily, and mice of group C, group D, and group E werefed with different freshly prepared probiotic solutions daily, that is,the Lactobacillus plantarum CMU995, the Lactobacillus rhamnosus GG, andthe Lactobacillus casei Shirota, respectively. Hepatitis induction wasstarted at day 3, and mice of group B, group C, group D, and group Ewere started to be fed with ethanol-containing Lieber-DeCarli liquiddiet. The concentration of ethanol of such an ethanol-containingLieber-DeCarli liquid diet ranges from 1.25% to 5%; the concentrationincreases 1.25% every two days till the highest concentration (5%).These mice received the highest ethanol concentration (5%) for a totalof six weeks, and the total duration of the hepatitis induction is sevenweeks.

(4) Animal Sacrifice and Sample Preparation

The day of the mice started to be fed with ethanol-containingLieber-DeCarli liquid diet is defined as day 0, and the mice weresacrificed after seven weeks (49 days). Appropriate amounts of bloodsamples of the mice were collected at day 0 and day 28 via facial veinin mice, and the mice were fasted one day before the day of sacrifice.The mice were sacrificed by cervical dislocation, and whole bloodsamples were collected. Liver samples and large intestine samples of themice were collected, frozen, and stored at −80° C.

(5) Blood, Liver, and Intestinal Samples Analysis

Appropriate amounts of frozen liver and intestinal samples wererespectively placed on a mortar and grinded with liquid nitrogen, andthen these samples were collected and mixed with appropriate amounts ofHEPES buffer for further analytical experiments. The blood samples werecentrifuged at low speed thereby isolating the blood serum for furtheranalytical experiments. Analysis of blood samples including aspartateaminotranferase (AST), alanine aminotransferase (ALT), trglyceride (TG),cholestrol, endotoxin, and cytokine TNF-α, were analyzed by using anautomated hematology analyzer (Cobas Mira Plus analyzer, Roche, USA)and/or enzyme-linked immunosorbent assay (ELISA).

(6) Statistical Analysis

Statistical analyses of the experimental results were performed usingStatistical Analysis System, V. 8.02 for Windows, 2001, with analysis ofvariance (ANOVA) test. A significant threshold of P<0.05 was employedfor the ANOVA analyses. The diagrams and figures performing theexperimental results were performed using SigmaPlot 10.0.

Animal Experiment Results

FIG. 1 and FIG. 2 are bar charts showing different levels of aspartateaminotransferase (AST) and alanine aminotransferase (ALT) in bloodserum, respectively, of mice of group A to group E according to theanimal experiment of the present disclosure. Levels of AST and ALT ofthe hepatitis-induced and Lactobacillus plantarum CMU995 fed mice (GroupC) were significantly lower than those of the group B, group D, andgroup E, which were also hepatitis induced. Therefore, the Lactobacillusplantarum CMU995 is capable of reducing the AST and ALT level inhepatitis-induced mice blood serum, so that it is capable of avoidingalcohol-caused liver damages, thereby protecting the liver.

FIG. 3 and FIG. 4 are bar charts showing different levels oftriglyceride and cholesterol in blood serum, respectively, of mice ofgroup A to group E according to the animal experiment of the presentdisclosure. Levels of triglyceride and cholesterol of thehepatitis-induced and Lactobacillus plantarum CMU995 fed mice (Group C)were significantly lower than those of the group B, group D, and groupE, which were also hepatitis induced. Therefore, the Lactobacillusplantarum CMU995 is capable of reducing the triglyceride and cholesterollevel in hepatitis-induced mice blood serum, so that it is capable ofavoiding alcohol-caused hyperlipidemia, thereby protecting the liver.

FIG. 5 and FIG. 6 are bar charts showing different levels of endotoxinin blood serum and liver, respectively, of mice of group A to group Eaccording to the animal experiment of the present disclosure. Levels ofblood serum endotoxin and liver endotoxin of the hepatitis-induced andLactobacillus plantarum CMU995 fed mice (Group C) were significantlylower than those of the group B, group D, and group E, which were alsohepatitis induced. Therefore, the Lactobacillus plantarum CMU995 iscapable of reducing the blood serum endotoxin and liver endotoxin levelin hepatitis-induced mice, so that capable of avoiding entries ofalcohol-induced endotoxin into blood, thereby protecting the liver.

FIG. 7 and FIG. 8 are bar charts showing different levels of liver andlarge intestinal cytokine TNF-α, respectively, of mice of group A togroup E according to the animal experiment of the present disclosure.Levels of liver and intestinal cytokine TNF-α of the hepatitis-inducedand Lactobacillus plantarum CMU995 fed mice (Group C) were significantlylower than those of the group B, group D, and group E, which were alsohepatitis induced. Therefore, the Lactobacillus plantarum CMU995 iscapable of reducing the liver and intestinal cytokine TNF-α level inhepatitis-induced mice, so that it is capable of reducing inflammationof liver and intestine.

III. Clinic

In this clinical trial, 26 patients were included. Blood samples fromeach patient were collected before and after 4-week oral administrationof a probiotic composition containing the Lactobacillus plantarum CMU995for further analysis.

(1) Exclusion and Inclusion Conditions of Patients

Patients in this clinical trial should have met all the followingcriteria for inclusion: with confirmed cirrhosis, age between 30-70years old.

Patients were excluded from this trial if they met any one of thefollowing conditions:

1. Those with underling portal hypertension, such as hepatorenalsyndrome, or bacterial peritonitis.

2. Those with neurological disease and acute metabolic disease, such asstroke, cardiac failure, respiratory failure, acute renal failure, orichorrhemia.

3. Those with stage 3 to stage 4 severe hepatic coma.

4. Those with systemic disorders such as cancers, cardiopulmonarydysfunction, renal insufficiency.

5 Those who had been treated with probiotics related compositions orproducts.

6. Women who are pregnant or may possibly become pregnant or arelactating under a positive urine pregnant test.

(2) Probiotic Composition

The probiotic composition used in this clinical trial contains theLactobacillus plantarum CMU995 and other excipients. Each unit of theaforementioned probiotic composition contains 3 g powder including3×10¹⁰ CFU Lactobacillus plantarum CMU995, so that the concentration ofLactobacillus plantarum CMU995 of each unit of the probiotic compositionis 1×10¹⁰ CFU/g.

(3) Sample Analysis

26 patients were participated in the clinical trial and had signed aconsent form. An amount of the probiotic composition for the 4-weekclinical trial was provided for the patients, and each of the patientswas treated with one unit of the probiotic composition after each meal.Blood samples of the patients were collected after 4 weeks for analysisincluding levels of cytokine TNF-α, endotoxin, and blood ammonia, inorder to evaluate the effects caused by probiotic composition. Thestatistical analyses of the results were performed by using SigmaPiot10.0.

Results

FIG. 9 a to FIG. 9 c are bar charts showing different levels of cytokineTNF-α, blood ammonia, and endotoxin, respectively, of the blood samplesfrom the cirrhosis patients who had been treated with the probioticcomposition. After treating the patients with the probiotic compositionfor 4 weeks, the levels of cytokine TNF-α, blood ammonia, and endotoxinsignificantly decreased (letter “a” differs from letter “b” means thatthe results before and after probiotic composition treatments arestatistically significant, that is, p<0.05). Therefore, it had beenproven that, by treating cirrhosis patients with the probioticcomposition containing the Lactobacillus plantarum CMU995 of the presentdisclosure, blood quality of the cirrhosis patients have beensignificantly improved.

Advantages of the Lactobacillus plantarum CMU995 of the presentdisclosure are described as follows:

1 The Lactobacillus plantarum CMU995 of the present disclosure is saltand acid tolerant, and can be well-adhered to intestinal epithelialcells of an animal host, so that the Lactobacillus plantarum CMU995 cansurvive in gastrointestinal system permanently. Therefore, theLactobacillus plantarum CMU995 may competitively inhibit pathogensadhere to the intestinal epithelial cells. Furthermore, referring to theaforementioned results of the adhesion analysis, the clinical trial, andthe animal experiment, the Lactobacillus plantarum CMU995 is capable ofpreventing damages of cells caused by exotoxins or endotoxins, therebystabilizing the intestinal epithelial cells. The Lactobacillus plantarumCMU995 is also capable of blocking alcohol (ethanol) and/or toxinspassing through intestines and then invading into blood.

2. For preventive use, the Lactobacillus plantarum CMU995 can besupplied in a form of a pharmaceutical composition, a feed, a drink, anutrient supplement, a diary, a food, or a healthy food, so that theLactobacillus plantarum CMU995 can be adhered to intestinal epithelialcells thereby blocking endotoxins, alcohol, or pathogens invading intoblood, and thus providing effects of protecting the liver, reducingblood endotoxin, blood alcohol, and so on.

3. For therapeutic use, Lactobacillus plantarum CMU995-containing drugsor foods can be administered to those who had been invaded by pathogens,alcohol, or other toxins which cause diseases, so that pathogens,alcohol, or other toxins can be effectively reduced, or even removed.For example, liver cancer or hepatitis patients can be treated with theLactobacillus plantarum CMU995 for reducing phenomena of hyperamnionema,liver inflammation, or intestinal inflammation. In short, theLactobacillus plantarum CMU995 is capable of preventing or curinggastrointestinal diseases caused by toxins or alcohol.

4. The Lactobacillus plantarum CMU995 not only can inhibit intestinaltoxins invading into blood by stabilizing the intestinal epithelialcells, it may also reduce bacterial or toxin accumulation in intestines,and thus one who had intaken the Lactobacillus plantarum CMU995 can beprotected by a synergetic effect of these aforementioned detoxmechanisms. The word “detox” means that to expel various toxins, andthese toxins are harmful substances, mycotoxins, and carcinogens, etc.,such as intestinal endotoxins, hemotoxins, intestinal pathogens,alcohol, hazardous medicines, chemical hazardous substances, DEHP, etc.These toxins may invade into blood and transfer to other organs in ananimal or a human body, and thus damages of organs such as liver will becaused. The Lactobacillus plantarum CMU995 and compositions containingthe Lactobacillus plantarum CMU995 are also capable of accelerating theremoval of intestinal or blood harmful substances, pathogens, or alcoholfrom body.

5. Because that the Lactobacillus plantarum CMU995 is able to inhibittoxins invade into blood, and to accelerate the removal of toxins, it isable to be widely applied to various medical and health relatedproducts. Therefore, a probiotic composition which contains theLactobacillus plantarum CMU995 is also provided in the presentdisclosure. This probiotic composition can be used to inhibit absorptionof various types of toxins, alcohol, and can also be used to inhibitpathogens by competitive inhibition.

6. The Lactobacillus plantarum CMU995 can be used to inhibit variousgastrointestinal harmful substances such as toxins released frombacteria, toxins released from fungi, toxins released from viruses,toxins released from foods, alcohol, pesticides, drugs, food additives,chemical hazardous substances, and DEHP, especially alcohol andmicroorganisms released toxins.

7. The probiotic composition which contains the Lactobacillus plantarumCMU995 of the present disclosure can be in the form of a pharmaceuticalcomposition, a feed, a drink, a nutrient supplement, a diary, a food, ora healthy food. Besides, the Lactobacillus plantarum CMU995 can be alsoadded to various kinds of food, such as dairy products, etc.

8. Further, addictives can also be added to the probiotic composition ofthe present disclosure for enhancing the adherent efficiency of theLactobacillus plantarum CMU995, thereby enhancing prevention of toxinsinvading into blood, and accelerating removal of toxins Nutrientsupplements and/or pharmaceutical supplements may also be added to thisprobiotic composition to increase the range of its use. For example,kinds of vitamins, Chinese herbal medicines, or other probiotics, etc.,can be added to this probiotic composition, to enhance the removal oftoxins or liver metabolism. Any addictives without negative effects canbe added and mixed with the probiotic composition to perform synergeticeffects of those.

9. The Lactobacillus plantarum CMU995 and the probiotic compositioncontaining the Lactobacillus plantarum CMU995 can be administered to ananimal or a human body in the form of a pharmaceutical composition, afeed, a drink, a nutrient supplement, a diary, a food, or a healthyfood, in order to protect intestinal epithelial cells, reduce intestinaltoxins, reduce blood toxins, reduce alcohol absorption, protect theliver and other organs in an animal or a human body, thereby preventingor curing diseases.

Although the present disclosure has been described in considerabledetail with reference to certain embodiments thereof, other embodimentsare possible. Therefore, the spirit and scope of the appended claimsshould not be limited to the description of the embodiments containedherein.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentdisclosure without departing from the scope or spirit of the invention.In view of the foregoing, it is intended that the present disclosurecover modifications and variations of this invention provided they failwithin the scope of the following claims.

What is claimed is:
 1. A probiotic composition for liver protectioncomprising powdered Lactobacillus plantarum CMU995, wherein theconcentration of the powdered Lactobacillus plantarum CMU995 is 1×10⁵CFU/g to 1×10¹² CFU/g, and the Lactobacillus plantarum CMU995 isdeposited at the Food Industry Research and Development Institute(FIRDI) in Taiwan under accession number BCRC 910472 and in the GermanCollection of Microorganisms and Cell Cultures (DSMZ) under accessionnumber DSM
 23780. 2. The probiotic composition according to claim 1,wherein the probiotic composition is in the form of a pharmaceuticalcomposition, a feed, a drink, a nutrient supplement, a diary, a food, ora healthy food.
 3. The probiotic composition according to claim 1,wherein the probiotic composition is used for treating hepatitis causedby alcohol and endotoxin, and reducing endotoxin-induced liver lesionsand/or hepatocarcinogenesis.
 4. The probiotic composition according toclaim 1 wherein the probiotic composition is in the form of a powder, atablet, a granule, a suppository, a capsule, an ampoule, a liquid, or aspray.